Project Summary The elderly population is rapidly increasing worldwide and those over 60 years old are expected to comprise 20% of the total population by 2050. These demographic changes will focus healthcare needs on those conditions more prevalent in the elderly. Chronic wounds, such as venous leg ulcers, pressure ulcers and diabetic ulcers are over-represented in the elderly population and even now present a significant healthcare burden. These conditions impact approximately 6.5 million patients annually with a cost to the health care system of over $25B. Rarely occurring in the young, the healing of these chronic wounds is confounded by age-related factors such as reduced blood flow, dermal atrophy, increased inflammation and reduced growth factor responses. The high recurrence rate and failure to respond to current therapies indicates the need for improved regenerative therapeutics that can overcome many of the age-associated defects in the wound healing process. Stem cell therapies represent a compelling means of tissue repair and have demonstrated wound healing and soft tissue regeneration in animal models. However, the intuitive concept that therapeutic stem cells engraft and differentiate at sites of tissue damage is not well supported given the low numbers of cells retained over time at application sites in vivo. This suggests that their mechanisms of action occur through paracrine modalities such as secretion of bioactive vesicles, including exosomes. Hence, exploiting stem cell-derived exosomes as a biologic-derived therapy, rather than delivering transient stem cells to treat chronic wounds, is an enticing approach. Secreted extracellular vesicles (EVs), such as exosomes, are packed with potent pro-repair proteins and RNA cargos that are both cell type- specific, as well as, differentially produced and secreted according to the cellular environment. Based on our compelling preliminary data, this Phase I proof-of-concept study will demonstrate that critical ex vivo environmental cues can tune stem cell-derived EVs to be pro-healing and that pro-healing EVs are a valid chronic wound therapeutic. We will achieve these goals by the following Aims: 1) manipulate the bioreactor environment to generate differential extracellular vesicle packaging, 2) in vitro testing of EV-mediated regenerative activities in fibroblasts, keratinocytes and endothelial cells, and 3) in vivo testing of pro-healing EVs in chronic wound healing models. This Phase I research program is designed to provide critical proof-of-concept data demonstrating manufacturing conditions and wound healing properties of stem cell- derived extracellular vesicles. This potential therapeutic has the unique advantage of harnessing the power of stem cells without the need for utilizing complex cell therapies in vivo.